Exposure to exogenous estrogen used in oral contraceptives (OCs) and hormone therapy (HT) is a major risk factor for breast cancer initiation and progression in women including veteran population. The signaling mechanisms by which estrogen contributes in the initiation or the progression of breast cancer are not fully understood. Understanding the exact molecular mechanisms by which breast cancer lesions arise upon chronic estrogen exposure could have major clinical implications for preventing or circumventing the disease. The overall goal of this proposal is to test a new paradigm that reactive oxygen species (ROS)-mediated activation of NRF-1 may contribute to predisposing veteran and civilian women to estrogen-induced breast cancer. This paradigm will be tested using: state of the art molecular tools, in vitro 2-D/3-D cell models, [human breast tissue specimens] and in vivo xenograft model. We and others have found that ROS are important signaling molecules, which contribute in the development of estrogen-induced malignant breast lesions. However, the molecular signaling mechanism by which ROS contribute to breast cancer susceptibility is not clear. Recent findings suggest the existence of other mechanism(s) independent of ER status that mediate estrogen- induced cell signaling leading to malignant transformation and growth of breast cancer cells. One such mechanism being investigated by us is the role of estrogen-induced ROS in breast carcinogenesis. Recently, we have observed that (i) estrogen-induced breast tumor formation in the xenograft model was inhibited by overexpression of the antioxidant enzyme, catalase, that lowers the level of H2O2 or by co- treatment with a chemical antioxidant, ebselen, that scavenges ROS; and (ii) NRF-1 overexpression supported tumor formation while NRF-1 knockdown reduced tumor formation. These findings suggest that ROS-induced NRF-1 activation may be a determinant of susceptibility to estrogen-induced breast cancer. Hypothesis: The above observations form the foundation of our hypothesis that ROS promotes susceptibility to estrogen-induced breast carcinogenesis by affecting the redox sensitive protein NRF-1 because modulation of NRF-1 affects breast tumor formation. Objectives: To address the above hypothesis, we propose three Specific Aims: (1) To determine that estrogen-induced ROS are essential for phosphorylation and acetylation of NRF-1. (2) To determine that estrogen-induced ROS control the transcription of target genes by (i) phosphorylation and acetylation of the NRF-1 transcription factor, and (ii) chromatin modifications via DNA oxidation and histone acetylation. (3) To determine whether pharmacological or genetic disruption of ROS and/or NRF-1 prevents the development of estrogen-induced mammary tumors. Procedures to be used: The study will be conducted using in vitro 2-D and 3-D cell culture models and findings will be further validated [in human breast tissue specimens] and in vivo by the xenograft nude mice model. This study will employ state of the art biochemical and molecular techniques, including gene silencing, immunoprecipitation, Western blotting, confocal imaging, ChIP, Real-Time RT-PCR, and cell transformation assays. Significance of potential new findings: The results of our proposed studies will elucidate, for the first time, the molecular mechanism by which estrogen-induced ROS regulates phosphorylation and acetylation of the transcription factor NRF-1 and define the role of NRF-1 signaling in estrogen-induced breast cancer pathogenesis that may prove useful therapeutic targets for the prevention and treatment of breast cancer. Furthermore, the findings from our study may set the stage to assess if phosphorylated and/or acetylated NRF-1 can serve as a valuable biomarker for breast cancer diagnosis and prognosis.